RT Journal Article SR Electronic T1 Deorphanization of GPRC6A: A Promiscuous l-α-Amino Acid Receptor with Preference for Basic Amino Acids JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 589 OP 597 DO 10.1124/mol.104.007559 VO 67 IS 3 A1 Wellendorph, Petrine A1 Hansen, Kasper B. A1 Balsgaard, Anders A1 Greenwood, Jeremy R. A1 Egebjerg, Jan A1 Bräuner-Osborne, Hans YR 2005 UL http://molpharm.aspetjournals.org/content/67/3/589.abstract AB One of the most important tasks of molecular pharmacology is the deorphanization of the large number of G-protein-coupled receptors with unidentified endogenous agonists. We recently reported the cloning and analysis of expression of a novel human family C G-protein-coupled receptor, termed hGPRC6A. To identify agonists at this orphan receptor, we faced the challenges of achieving surface expression in mammalian cell lines and establishing an appropriate functional assay. Generating a chimeric receptor construct, h6A/5.24, containing the ligand binding amino-terminal domain (ATD) of hGPRC6A with the signal transducing transmembrane and C terminus of the homologous goldfish 5.24 receptor allowed us to overcome these obstacles. Homology modeling of the hGPRC6A ATD based on the crystal structure of the metabotropic glutamate receptor subtype 1 predicted interaction with α-amino acids and was employed to rationally select potential ligands. Measurement of Ca2+-dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of h6A/5.24 and identification of l-α-amino acids as agonists. The most active agonists were basic l-α-amino acids, l-Arg, l-Lys, and l-ornithine, suggesting that these may function as endogenous signaling molecules. Measurement of intracellular calcium in tsA cells expressing h6A/5.24 allowed determination of EC50 values, which confirmed the agonist preferences observed in oocytes. Cloning, cell surface expression and deorphanization of the mouse ortholog further reinforces the assignment of the agonist preferences of hGPRC6A. This study demonstrates the utility of a chimeric receptor approach in combination with molecular modeling, for elucidating agonist interaction with GPRC6A, a novel family C G-protein-coupled receptor.